US7842477B2 - Methods for producing gamma-carboxylated proteins - Google Patents

Methods for producing gamma-carboxylated proteins Download PDF

Info

Publication number
US7842477B2
US7842477B2 US10/964,888 US96488804A US7842477B2 US 7842477 B2 US7842477 B2 US 7842477B2 US 96488804 A US96488804 A US 96488804A US 7842477 B2 US7842477 B2 US 7842477B2
Authority
US
United States
Prior art keywords
protein
promoter
cell
gamma
carboxylation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related, expires
Application number
US10/964,888
Other languages
English (en)
Other versions
US20050164367A1 (en
Inventor
Christel Fenge
Ann Lövgren
Anders Thelin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MedImmune Ltd
Original Assignee
AstraZeneca AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=29559260&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US7842477(B2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by AstraZeneca AB filed Critical AstraZeneca AB
Assigned to ASTRAZENECA AB reassignment ASTRAZENECA AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: THELIN, ANDERS, LOVGREN, ANN, FENGE, CHRISTEL
Publication of US20050164367A1 publication Critical patent/US20050164367A1/en
Priority to US12/950,713 priority Critical patent/US8697440B2/en
Application granted granted Critical
Publication of US7842477B2 publication Critical patent/US7842477B2/en
Assigned to MEDIMMUNE LIMITED reassignment MEDIMMUNE LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ASTRAZENECA AB
Expired - Fee Related legal-status Critical Current
Adjusted expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/647—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences comprising a first promoter and a nucleic acid molecule encoding a ⁇ -glutamyl carboxylase and associated expression control sequences comprising a second promoter.
  • the invention further relates to a method of producing a protein requiring gamma-carboxylation in high yields.
  • Bleeding is a common clinical problem. It is a consequence of disease, trauma, surgery and medicinal treatment. It is imperative to mechanically stop the bleeding. This may be difficult or even impossible due to the location of the bleeding or because it diffuses from many (small) vessels. Patients who are bleeding may thus require treatment with agents that support haemostasis. This may be blood-derived products (haemotherapy), agents that cause the release of endogenous haemostatic agents, recombinant coagulation factors (F), or agents that delay the dissolution of blood clots.
  • haemotherapy blood-derived products
  • F recombinant coagulation factors
  • the first line treatment among the blood derived products are whole blood for volume substitution and support of haemostasis, packed red cells for the improvement of oxygen transporting capacity, platelet concentrates to raise the number of platelets (if low or defective) and fresh frozen plasma for support of the haemostasis (blood coagulation and platelet aggregation).
  • Second line plasma derived products that support haemostasis are plasma cryoprecipitate, prothrombin complex concentrates, activated prothrombin complex concentrates and purified coagulation factors.
  • Several coagulation factors are today available as human recombinant proteins, inactive (coagulation factors VIII and IX) and activated (coagulation factor VIIa).
  • Haemophilia is an inherited or acquired bleeding disorder with either abnormal or deficient coagulation factor or antibodies directed towards a coagulation factor which inhibits the procoagulant function.
  • the most common haemophilias are haemophilia A (lack coagulation factor VIII) and haemophilia B (factor IX).
  • the purified or recombinant single coagulation factors are the main treatment of patients with haemophilia. Patients with inhibitory antibodies posses a treatment problem as they may also neutralise the coagulation factor that is administered to the patient.
  • the active form of Protein C (APC) is an inhibitor of plasma coagulation by degradation of the activated coagulation factors Va and VIIIa. Recombinant APC has been shown to be an effective treatment of undue plasma coagulation in patients with sepsis.
  • Coagulation factors for therapeutic use can be obtained from human plasma, although the purification process is not simple and requires many steps of which several aim at eliminating contaminating viruses. But even with extensive safety measures and testing of blood-derived products, contamination with infectious viruses or prions cannot be ruled out. Because of this risk it is highly desirable to produce human therapeutic proteins from recombinant cells grown in media without animal derived components. This is not always straightforward as many proteins require a mammalian host to be produced in a fully functional form, i.e. be correctly post-translationally modified.
  • coagulation factors commercially produced in recombinant cells are FVII (NovoSeven), FVIII (Kogenate, Recombinate, Refacto) and FIX (BeneFix) (Roddie and Ludlam. Blood Rev. 11: 169-177, 1997) and Active Protein C (Xigris).
  • FVII NovoSeven
  • FVIII Kogenate, Recombinate, Refacto
  • FIX Boddie and Ludlam. Blood Rev. 11: 169-177, 1997)
  • Active Protein C Xigris
  • One of the major obstacles in obtaining large amounts of fully functional recombinant human coagulation factors lies in the Gla-domain present in FII, FVII, FIX, FX and Protein C. This domain contains glutamic acid residues that are post-translationally modified by addition of carboxyl groups.
  • Gla modifications are a result of the action of a vitamin K-dependent enzyme called ⁇ -glutamyl carboxylase (GGCX).
  • GGCX ⁇ -glutamyl carboxylase
  • prothrombin For human FII (prothrombin) at least 8 out of 10 Glu residues have to be correctly modified in order to obtain fully functional prothrombin (Malhotra, et al., J. Biol. Chem. 260: 279-287, 1985; Seegers and Walz ‘Prothrombin and other vitamin K proteins’, CRC Press, 1986). Extensive efforts to obtain high production levels of rhFII have been made using several different systems such as CHO cells, BHK cells, 293 cells and vaccinia virus expression systems, but have all failed or resulted in an under-carboxylated product and thus functionally inactive prothrombin (J ⁇ rgensen et al., J. Biol. Chem.
  • WO 92/19636 discloses the cloning and sequence identification of a human and bovine vitamin K dependent carboxylase. The application suggests co-expressing the vitamin K dependent carboxylase and a vitamin K dependent protein in a suitable host cell in order to prepare the vitamin K dependent protein. No co-expression of the carboxylase and vitamin K dependent protein is exemplified.
  • a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences comprising a first promoter and a nucleic acid molecule encoding a ⁇ -glutamyl carboxylase and associated expression control sequences comprising a second promoter, wherein the first promoter is sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the ⁇ -glutamyl carboxylase are expressed in a ratio of at least 10:1.
  • a cell which is engineered to express (i) a protein which requires gamma-carboxylation, and (ii) a ⁇ -glutamyl carboxylase, wherein the proteins (i) and (ii) are expressed in a ratio between 10:1 and 500:1.
  • genetically modified eukaryotic host cell comprising:
  • the cell is capable of expressing the ⁇ -glutamyl carboxylase protein and the protein requiring carboxylation in the ratio of at least 1:10.
  • a vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences comprising a first promoter and a nucleic acid molecule encoding a ⁇ -glutamyl carboxylase and associated expression control sequences comprising a second promoter, wherein the first promoter is sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the ⁇ -glutamyl carboxylase are expressed in a ratio of at least 10:1.
  • a method for producing gamma-carboxylated protein comprising: (i) culturing a cell adapted to express a protein which requires gamma-carboxylation and ⁇ -glutamyl carboxylase in a ratio of at least 10:1, under conditions suitable for expression of both proteins, and (ii) isolating gamma-carboxylated protein.
  • the method is used for producing gamma-carboxylated human Factor IX and in another embodiment the method is used for producing gamma-carboxylated human prothrombin.
  • the gamma-carboxylated protein produced is human gamma-carboxylated Factor X.
  • a method for production of a gamma-carboxylated protein in a mammalian cell line comprising the step of co-expressing with said protein requiring gamma-carboxylation in the mammalian cell line a ⁇ -glutamyl carboxylase, wherein the amount of expressed protein requiring gamma-carboxylation is at least 10-fold greater than the amount of expressed ⁇ -glutamyl carboxylase, and (ii) isolating gamma-carboxylated protein.
  • the method is used for producing gamma-carboxylated human Factor IX and in another embodiment the method is used for producing gamma-carboxylated human prothrombin.
  • the gamma-carboxylated protein produced is human gamma-carboxylated Factor X.
  • isolated gamma-carboxylated protein produced according to the above methods and the use of isolated gamma-carboxylated protein produced according to the above methods in coagulation therapy or the use of isolated gamma-carboxylated protein produced according to the above methods for the manufacture of a medicament for use in coagulation therapy.
  • a method of producing a pharmaceutical composition suitable for inducing blood clotting or promoting increased or decreased coagulation comprising purifying active carboxylated protein expressed from a host cell adapted to express a protein requiring gamma-carboxylation and ⁇ -glutamyl carboxylase in a ratio of between 10:1 and 500:1 and admixing the purified carboxylated protein with one or more pharmaceutically acceptable carriers or excipients and a pharmaceutical composition obtainable from the method.
  • the active carboxylated protein is gamma-carboxylated human Factor IX and in another embodiment the active carboxylated protein is gamma-carboxylated human prothrombin.
  • the active carboxylated protein is gamma-carboxylated Factor X.
  • FIG. 1 a illustrates a plasmid map of PN32 (prothrombin+GGCX) co-expression vector.
  • FIG. 1 b represents a plasmid map of Ptext5 (prothrombin) expression vector.
  • FIG. 2 represents a plasmid map of PP6 (prothrombin+GGCX) co-expression vector.
  • FIG. 3 a represents a plasmid map of F9NopA (factor IX+GGCX) co-expression vector
  • FIG. 3 b represents a plasmid map of F9hglx (factor IX+GGCX) co-expression vector.
  • the vitamin K dependent coagulation factors (FII, FVII, FIX, FX and their activated forms FIIa or thrombin, FVIIa, FIXa, FXa) produced by the present method of co-expression with GGCX can be expected to be useful in the prevention and treatment of bleeding following trauma, surgery or diseases of the liver, kidneys, platelets or blood coagulation factors (haemophilia).
  • the coagulation factor Protein C and its activated form APC can be expected to be useful in the prevention and treatment of disorders of increased coagulation with or without decreased levels of Protein C.
  • the method is also applicable to other proteins that require post-translational carboxylation.
  • a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences comprising a first promoter and a nucleic acid molecule encoding a ⁇ -glutamyl carboxylase and associated expression control sequences comprising a second promoter, wherein the first promoter is sufficiently stronger than the second promoter so that the protein requiring gamma-carboxylation and the ⁇ -glutamyl carboxylase are expressed in a ratio of at least 10:1.
  • the ratio of the expressed proteins is between 10:1 and 1000:1, more preferably between 10:1 and 500:1 and still more preferably between 25:1 and 250:1.
  • a particularly suitable ratio is around 200:1.
  • the ratio of the two expressed proteins can be at least 10:1, 30:1, 45:1, 50:1, 100:1, 200:1, 250:1, 300:1, 400:1, 500:1 and 1000:1.
  • both the nucleic acid molecule encoding the protein requiring gamma-carboxylation and associated expression control sequences, and the nucleic acid molecule encoding the ⁇ -glutamyl carboxylase and associated expression control sequences are located on the same expression vector. In another embodiment these two nucleic acid molecules are located on separate expression vectors.
  • nucleic acid according to SEQ ID NO: 14 and SEQ ID NO: 15.
  • host cells transfected or transformed with a vector comprising the sequence of SEQ. ID NO: 14 or SEQ ID NO: 15 for the expression of human Factor IX.
  • a non-human eukaryotic host cell adapted to express human coagulation factor IX and human gamma carboxylase enzymes in a ratio of at least 10:1.
  • the nucleic acid encoding the human coagulation factor IX and the nucleic acid encoding the gamma carboxylase are operably linked to control sequences that are capable of expressing the two proteins in a ratio of at least 10:1, respectively.
  • a host cell harbouring exogenous nucleic acid comprising human coagulation factor IX encoding nucleic acid under the control of hCMV promoter and human carboxylase encoding nucleic acid under the control of SV40 promoter.
  • host cells transfected or transformed with a vector comprising the sequence of SEQ. ID NO: 1, SEQ. ID NO: 2 or SEQ ID NO: 3 for the expression of human prothrombin.
  • host cells capable of expressing human prothrombin and human gamma carboxylase enzymes, wherein the nucleic acid encoding the human prothrombin and the nucleic acid encoding the gamma carboxylase are operably linked to control sequences that are capable of expressing the two proteins in a ratio of at least 10:1, respectively.
  • a non-human eukaryotic host cell adapted to express human prothrombin and human gamma carboxylase enzymes in a ratio of at least 10:1.
  • the nucleic acid encoding the human prothrombin and the nucleic acid encoding the gamma carboxylase are operably linked to control sequences that are capable of expressing the two proteins in a ratio of at least 10:1, respectively.
  • the invention has been exemplified using prothrombin and coagulation factor IX as the proteins requiring carboxylation.
  • prothrombin and factor IX are dependent on correct ⁇ -carboxylation for their full biological activity.
  • coagulation factor FVII which at present is only commercially produced in recombinant mammalian cells at relatively low levels (approximately 10 mg/L or less).
  • the present invention will be applicable to improve the productivity of any protein that is dependent on ⁇ -carboxylation
  • proteins include, but are not limited to: prothrombin, coagulation factor II (FII), coagulation factor VII (FVII), coagulation factor IX (FIX), coagulation factor X (FX), Protein C, Protein S, Protein Z, Bone Gla protein (also known as: BGP or osteocalcin), Matrix Gla protein (MGP), proline rich Gla polypeptide 1 (PRRG1), proline rich Gla polypeptide 2 (PRRG2), Growth arrest-specific protein 6 (Gas 6).
  • Other suitable proteins are: FXa-like protein in venom of elapid snake (subfamily Acanthophiina) and cone snail venom ( Conus textile ).
  • the invention is not restricted to a particular protein or protein encoding sequence of one of these proteins to be co-expressed. Moreover, and in particular with respect to blood coagulation factors, numerous mutant forms of the proteins have been disclosed in the art. The present invention is equally applicable to these mutant forms, including naturally occurring allelic variants, of the proteins as it is to wild-type sequence. In one embodiment the invention can be undertaking with any wild-type protein or one with at least 90%, preferably at least 95% sequence identity thereto.
  • sequence identity between two sequences can be determined by pair-wise computer alignment analysis, using programs such as, BestFit, PILEUP, Gap or FrameAlign.
  • the preferred alignment tool is BestFit.
  • suitable algorithms such as Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, or Fasta3, and a scoring matrix such as Blosum 62.
  • Such algorithms endeavour to closely approximate the “gold-standard” alignment algorithm of Smith-Waterman.
  • the preferred software/search engine program for use in assessing similarity i.e., how two primary polypeptide sequences line up is Smith-Waterman. Identity refers to direct matches, similarity allows for conservative substitutions.
  • ⁇ -glutamyl carboxylase or “GGCX”, as used herein, refers to a vitamin K dependent enzyme that catalyses carboxylation of glutamic acid residues.
  • GGCX enzymes are widely distributed, and have been cloned from many different species such as the beluga whale Delphinaptrus leucas , the toadfish Opsanus tau , chicken ( Gallus gallus ), hagfish ( Myxine glutinosa ), horseshoe crab ( Limulus polyphemus ), and the cone snail Conus textile (Begley et al., 2000, ibid; Bandyopadhyay et al. 2002, ibid).
  • the carboxylase from conus snail is similar to bovine carboxylase and has been expressed in COS cells (Czerwiec et al. 2002, ibid).
  • GGCX GGCX-like proteins similar to GGCX can be found in insects and prokaryotes such as Anopheles gambiae, Drosophila melanogaster and Leptospira with NCBI accession numbers: gi 31217234, gi 21298685, gi 24216281, gi 24197548 and (Bandyopadhyay et al., 2002, ibid), respectively.
  • the carboxylase enzyme displays remarkable evolutionary conservation.
  • Several of the non-human enzymes have shown, or may be predicted to have, activity similar to that of the human GGCX we have used, and may therefore be used as an alternative to the human enzyme.
  • GGCX proteins and GGCX proteins from other species can be used as the carboxylase enzyme in the present invention.
  • One way to effect differential expression of the two co-expressed proteins is to use different promoters as part of the respective expression control sequences.
  • the art is replete with examples of different promoters and other expression control sequences that are capable of expressing heterologous proteins to differing degrees or extents.
  • Recombinant expression technology is suitably advanced such that a person skilled in the art of protein expression is able to select promoters and other control sequences to bring about co-expression of the protein requiring carboxylation and the carboxylase in the desired ratio.
  • the selection of which particular promoters and other expression control sequences to use is a matter of individual choice.
  • control sequences associated with the protein requiring gamma-carboxylation comprise a strong promoter.
  • this is the human cytomegalovirus (hCMV) immediate-early promoter.
  • hCMV human cytomegalovirus
  • a strong promoter is here defined as a promoter giving rise to more than 1000 transcripts/cell.
  • a weak promoter is here defined as a promoter giving rise to less than 1000 transcripts/cell.
  • control sequences associated with the ⁇ -glutamyl carboxylase comprise a weak promoter. In one embodiment this is SV40 early promoter.
  • protein requiring gamma-carboxylation and the ⁇ -glutamyl carboxylase are under the control of different promoter elements with the promoter controlling expression of the ⁇ -glutamyl carboxylase being weaker that the promoter controlling expression of the protein requiring gamma-carboxylation.
  • the ⁇ -glutamyl carboxylase is under the control of SV40 early promoter and the protein requiring gamma-carboxylation is under the control of the human cytomegalovirus (hCMV) immediate-early promoter.
  • the protein requiring gamma-carboxylation is human Factor X.
  • the protein requiring gamma-carboxylation is human prothrombin.
  • the protein requiring gamma-carboxylation is human Factor IX.
  • the invention has been exemplified by use of the strong CMV promoter (Boshart et al. Cell 41: 521-530, 1985) to over-express Factor 1 ⁇ or prothrombin and the weaker SV40 promoter (Wenger et al. Anal Biochem 221: 416-418, 1994) to control the GGCX expression.
  • pEF-1 ⁇ human elongation factor-1 ⁇ subunit gene
  • pRSV Ratio-1 sarcoma virus
  • pUbC human ubiquitin
  • a cell which is engineered or adapted to express (i) a protein which requires gamma-carboxylation, and (ii) a ⁇ -glutamyl carboxylase, wherein the proteins (i) and (ii) are expressed in a ratio between 10:1 and 500:1.
  • the ⁇ -glutamyl carboxylase is expressed between 2 and 5-fold above endogenous levels (i.e. that in a non-engineered or adapted cell).
  • a recombinant cell adapted to express (i) ⁇ -glutamyl carboxylase protein above constitutive levels found in an equivalent unadapted cell and (ii) a protein requiring carboxylation, wherein the amount of expressed ⁇ -glutamyl carboxylase protein and protein requiring carboxylation is in the ratio of at least 1:10.
  • a genetically modified eukaryotic host cell comprising:
  • the cell is capable of expressing the ⁇ -glutamyl carboxylase protein and the protein requiring carboxylation in the ratio of at least 1:10.
  • a cell adapted to express a protein which requires gamma-carboxylation and ⁇ -glutamyl carboxylase, wherein the nucleic acid encoding the protein which requires gamma-carboxylation and the nucleic acid encoding the ⁇ -glutamyl carboxylase are under the control of regulatory sequences suitable for ensuring that the amount of expressed protein which requires gamma-carboxylation is at least 10-fold the amount of the ⁇ -glutamyl carboxylase protein.
  • At least one of the protein which requires gamma-carboxylation and the ⁇ -glutamyl carboxylase is expressed from nucleic acid that has been introduced into the cell by recombinant technology.
  • An alternate way of working the invention is to express endogenous protein (protein requiring carboxylation or carboxylase), but with substitution of the endogenous control sequences (promoter etc.) with heterologous sequences to effect the desired level of expression.
  • the host cell is preferably a eukaryotic cell.
  • Typical host cells include, but are not limited to insect cells, yeast cells, and mammalian cells. Mammalian cells are particularly preferred. Suitable mammalian cells lines include, but are not limited to, CHO, HEK, NSO, 293, Per C.6, BHK and COS cells, and derivatives thereof.
  • the host cell is the mammalian cell line CHO-S.
  • a cell or cell line which has little or no constitutively expressed carboxylase and/or protein requiring carboxylation.
  • the degree expression of the two proteins can be measured using techniques familiar to a person skilled in the art. These include direct measurements, for example measuring biological activity of the protein, or amount of protein (e.g. using antibodies), or indirect measurements, for example via measurement of mRNA transcript levels (e.g. Taqman analysis as in Example 3).
  • the ratio of expression of the two proteins is determined indirectly via mRNA transcript level (e.g., by Taqman analysis).
  • the present invention has general application to proteins that require carboxylation from any source. However, if the expressed protein is to be used for human therapeutic purposes, human proteins are particularly preferred.
  • a method for producing gamma-carboxylated protein comprising: (i) culturing a cell adapted to express a protein which requires gamma-carboxylation and ⁇ -glutamyl carboxylase in a ratio of at least 10:1, under conditions suitable for expression of both proteins, and (ii) isolating gamma-carboxylated protein.
  • a method for production of a gamma-carboxylated protein in a mammalian cell line comprising the step of co-expressing with said protein requiring gamma-carboxylation in the mammalian cell line a ⁇ -glutamyl carboxylase, wherein the amount of expressed protein requiring gamma-carboxylation is at least 10-fold greater than the amount of expressed ⁇ -glutamyl carboxylase; and (ii) isolating gamma-carboxylated protein.
  • Expression vectors usually include an origin of replication, a promoter, a translation initiation site, optionally a signal peptide, a polyadenylation site, and a transcription termination site. These vectors also usually contain one or more antibiotic resistance marker gene(s) for selection. Suitable expression vectors may be plasmids, cosmids or viruses such as phage or retroviruses. The coding sequence of the polypeptide is placed under the control of an appropriate promoter (i.e., HSV, CMV, TK, RSV, SV40 etc), control elements and transcription terminator (these are the associated expression control sequences) so that the nucleic acid sequence encoding the polypeptide is transcribed into RNA in the host cell transformed or transfected by the expression vector construct.
  • an appropriate promoter i.e., HSV, CMV, TK, RSV, SV40 etc
  • the host cell can be genetically modified (have extra nucleic acids introduced) by numerous methods, well known to a person skilled in the art, such as transfection, transformation and electroporation.
  • the invention also extends to purified gamma carboxylated protein produced by the methods of the present invention and their use in coagulant therapy.
  • a method of promoting increased or decreased coagulation in a subject comprising administering a pharmacologically effective amount of an isolated gamma-carboxylated protein obtained by the above-described methods to a patient in need thereof.
  • a method of producing a pharmaceutical composition suitable for inducing blood clotting comprising purifying active carboxylated protein expressed from a host cell adapted to express a protein requiring gamma-carboxylation and ⁇ -glutamyl carboxylase in a ratio of at least 10:1 and admixing the purified carboxylated protein with one or more pharmaceutically acceptable carriers or excipients.
  • Protein-based therapeutics are usually stored frozen, refrigerated, at room temperature, and/or or in a freeze-dried state.
  • the aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
  • preservatives such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
  • the pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these.
  • Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate.
  • compositions of the invention may also be in the form of a sterile solution or suspension in a non-toxic parenterally acceptable diluent or solvent, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above.
  • a sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
  • the amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration.
  • a formulation intended for injection to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.
  • Dosage unit forms will generally contain about 1 mg to about 500 mg of the active ingredient. Proteinaceous therapeutics are usually stored frozen or freeze-dried.
  • the size of the dose for therapeutic or prophylactic purposes of a compound will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.
  • a daily dose in the range for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses.
  • lower doses will be administered when a parenteral route is employed.
  • a dose in the range for example, 0.5 mg to 30 mg per kg body weight will generally be used.
  • a dose in the range for example, 0.5 mg to 25 mg per kg body weight will be used.
  • Human liver mRNA was purchased from Clontech and cDNA synthesis was performed using the Superscript system from Invitrogen. The obtained cDNA was used as template for amplification of human FII using:
  • primer PTF0 (SEQ ID NO: 3) 5′-ATTCCTCAGTGACCCAGGAGCTGACA-3′, and primer PTEXT (SEQ ID NO: 4). 5′-CTACTCTCCAAACTGATCAATGACCTTCTGTATCCACTTCTT-3′, Human GGCX was Amplified Using
  • the FII-encoding PCR product was cloned directly into the TA-TOPO treated vector pCDNA3.1V5/His (Invitrogen). Selection of a clone with hFII cDNA inserted in the correct direction gave the Ptext5 control construct ( FIG. 1 b ).
  • GGCX encoding cDNA under the control of the SV40 promoter was obtained by transfer of the GGCX encoding fragment from pCDNA3.1V5/His TA-TOPO to the pZeoSV2+ vector (Invitrogen), using restriction enzymes BamH1 and NotI. The EcoRV-NotI restriction sites downstream of the GGCX insert were removed.
  • a blunted ClaI-BclI fragment from the resulting pZeoSV2-GGCX plasmid (containing the SV40 promoter and the GGCX containing insert, but not the polyadenylation site and polyadenylation signal downstream of the GGCX encoding sequence) was then cloned into the blunted DraIII restriction site of pCDNA3.1+(Invitrogen).
  • a clone with the pSV40-GGCX fragment inserted in tandem (same transcriptional direction) relative to the CMV promoter was selected and a blunted KpnI-NotI FII encoding fragment from Ptext5 was cloned into the EcoRV site to obtain the PN32 construct ( FIG. 1 a ).
  • the DNA sequences of PN32 and Ptext5 are as in Appendix 2. All cloning methods were according to standard methods and/or manufacturers' recommended procedures.
  • the PN32 construct contains the following key features:
  • FIG. 1 b For comparison the PText5 construct without GGCX was used ( FIG. 1 b ).
  • PTEXT5 nucleotide sequence is shown in SEQ ID NO: 1.
  • PN32 nucleotide sequence is shown in SEQ ID NO: 2.
  • the two constructs in FIG. 1 were transfected into CHO-S cells (Invitrogen). Stable transfectants were selected and screened for highly productive clones using a commercially available assay for prothrombin activity (Chromogenix). In this assay the prothrombin containing samples were first treated with snake venom toxin (Ecarin—available from Sigma) to generate thrombin. Thrombin activity was then assayed by addition of a chromogenic substrate (S-2238)—which generates colour when processed by thrombin. Transfection and selection of clones were done in parallel with both constructs. Cell culturing was done in DMEM medium containing 9% heat inactivated fetal bovine serum.
  • Clones obtained were then adapted to growth in animal component free medium.
  • the best producing clone obtained was from transfection with PN32 (FII+GGCX), which yielded up to 400 mg/L human recombinant prothrombin when grown in animal component free chemically defined medium (far in excess of any published levels).
  • Recombinantly produced rhFII was purified (according to the method disclosed in Josic et al., Journal of Chromatography B, 790: 183-197, 2003), and fractionated by ion-exchange chromatography using a Q-Sepharose column according to standard techniques, to obtain pure fully-carboxylated rhFII.
  • Two rhFII producing CHO-S cell lines obtained by transfection with the PN32 construct (co-expression of human GGCX) and PTEXT5 (no co-expression of GGCX), respectively, were grown in spinner bottles using protein free medium supplemented with 5 ⁇ g/ml vitamin K. One tenth of the growth medium was replaced daily. Cells were harvested after 7 days of culture and microsomes were prepared as described by Berkner et al., (Proc Natl Acad Sci USA 89: 6242-6246 1992). Human recombinant FII was purified from the culture supernate of the harvested cells.
  • GGCX activity was measured as described by Berkner and Pudota (Proc Natl Acad Sci USA 95: 446-471 1998; and, Lingenfelter and Berkner (Biochemistry 35: 8234-8243, 1996). Our measurements showed that the GGCX activity is 1.5 times higher in the human GGCX co-expressing CHO cell line compared to the CHO cell line expressing only rhFII, using the same growth conditions.
  • Primers were manufactured by Operon/Qiagen and the probes were ordered from Applied Biosystems. Rodent GAPDH control primers and probe were also used (Applied Biosystems; ABI # 4308318 TaqMan® Rodent GAPDH Control Reagents Protocol)-Amplicon length 177 bp.
  • the Real-Time RT-PCR reactions were performed on the ABI PrismTM 7700 Sequence detector, Applied Biosystems. The expected length of the amplified PCR products was confirmed on agarose gels. Dilution series to investigate the efficiency of the PCR reactions were carried out for all three genes. Expression levels of ⁇ -Carboxylase and Prothrombin are presented relative to the expression of the control gene, rodent GAPDH.
  • ratios of approximately 74-232:1 were calculated depending on day of sample collection.
  • the number of transcript per cell were calculated to be approximately 8 for the GGCX mRNA and approximately 2000 for the rhFII mRNA, thus giving a rhFII:GGCX ratio of approximately 250:1.
  • the GAPDH control mRNA transcripts/cell was for the same sample approximately 4000.
  • the human FII and GGCX cDNA cloned in Example 1 were inserted into pCDNA3.1 similarly as in Ex.1.
  • the polyadenylation signal from pZeoSV2+ was included in the pSV40-GGCX-pA fragment cloned into the blunted DraIII site of pCDNA3.1.
  • a clone with the GGCX-containing fragment in the reverse order compared to Ex. 1 was selected. Cloning of the FII fragment was then done the same way as in Ex 1.
  • the final construct PP6 is shown in FIG. 2 and the PP6 nucleotide sequence is shown in SEQ ID NO: 13.
  • prothrombin producing cell lines B2F4 and H3B10 were obtained by transfecting CHO-S as described in Ex 1. Prothrombin from these two cell lines was purified and characterized as in Ex 1. Cultures of B2F4 gave productivities ranging from 30-70 mg/L and the share of fully carboxylated from 55-87% (the more rhFII the less fully carboxylated). Addition of butyrate gave a somewhat higher productivity but decreased the share of fully carboxylated rhFII and was not considered to be beneficial. H3B10 is slow-growing and gave a productivity of about 50 mg/L, which was high relative to the amount of cells in the culture, and the share of fully carboxylated rhFII was around 60%. Compared to the cell line obtained in example 1, less fully carboxylated rhFII was produced using the PP6 construct for a CHO cell line. The production of fully active recombinant prothrombin is still, however, far above earlier published levels.
  • the B2F4 and H3B10 cell lines from example 4 were analysed by real-time PCR analyses by the same method and the same primers as in Ex 3. Culture samples of 10 ml were collected at peak productivity in order to be equivalent to samples in Ex 3. For clone H3B10 samples were from day 10 due to the slow growth of this clone, and for clone B2F4 samples were from day 6.
  • the calculated ratio rhFII mRNA: GGCX mRNA was approximately 30:1 for clone H3B10, approximately 50:1 for clone B2F4 and approximately 250:1 for clone P1E2.
  • Human coagulation factor IX cDNA was amplified from human Gene pool liver cDNA purchased from Invitrogen. Oligonucleotide primers were for
  • F9f.ampl. 5′-CACCATGCAGCGCGTGAACATGAT-3′, (SEQ ID NO: 16) and the 3′-end; F9r.ampl.: 5′-CCTTGGAAATCCATCTTTCATTA-3′. (SEQ ID NO: 17)
  • the vector construct F9NopA can be seen in FIG. 3 a and the vector construct F9hglx is shown in FIG. 3 b .
  • the difference between the vectors F9NopA and F9hglx is the transcription direction of the GGCX gene.
  • the F9NopA nucleotide sequence is shown in SEQ ID NO: 14 and the F9hglx nucleotide sequence is shown in SEQ ID NO: 15.
  • the rhFIX constructs were transfected to CHO-S cells using the procedure described in Ex1. For each FIX construct approximately 3000 clones were screened for rhFIX expression by ELISA of cell supernates. Antibodies used were from Haemathology Technology Inc. and DakoCytomation. Clones were selected and adapted to growth in protein free chemically defined CHO medium. Cells were grown either in T-flasks at 37° C. or in spinner bottles at 32-37° C. CO 2 concentration was 5% for both types of cultures. The rhFIX produced was purified to homogeneity by Q-Sepharose anion exchange chromathography at pH 7.0.
  • Recombinant hFIX activity was determined by Clotting assay using FIX deficient plasma (Precision Biologic).
  • the best producing rhFIX clone obtained was N4D5, which was obtained using the F9NopA construct, produced up to 4 ⁇ g/ml active rhFIX grown in protein free chemically defined medium in T-flask. Grown in spinner bottle the same clone produced up to 7.1 ⁇ g/ml rhFIX.
  • the productivity of the N4D5 cell line is approximately 4-6 better than previously published levels obtained under comparable conditions, wherein IC4, IG8, r-FIX BHK and r-FIX 293 is the name of the clones mentioned in the references (Table 5).
  • RNA levels were found to peak at different days depending on culture temperature and culture inoculum size. Peak levels of mRNA were found to correspond well with peak concentration of rhFIX in the culture medium.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Diabetes (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
US10/964,888 2003-10-14 2004-10-14 Methods for producing gamma-carboxylated proteins Expired - Fee Related US7842477B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/950,713 US8697440B2 (en) 2003-10-14 2010-11-19 Compositions and methods for producing gamma-carboxylated proteins

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0324044.7 2003-10-14
GBGB0324044.7A GB0324044D0 (en) 2003-10-14 2003-10-14 Protein

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/950,713 Continuation US8697440B2 (en) 2003-10-14 2010-11-19 Compositions and methods for producing gamma-carboxylated proteins

Publications (2)

Publication Number Publication Date
US20050164367A1 US20050164367A1 (en) 2005-07-28
US7842477B2 true US7842477B2 (en) 2010-11-30

Family

ID=29559260

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/964,888 Expired - Fee Related US7842477B2 (en) 2003-10-14 2004-10-14 Methods for producing gamma-carboxylated proteins
US12/950,713 Expired - Fee Related US8697440B2 (en) 2003-10-14 2010-11-19 Compositions and methods for producing gamma-carboxylated proteins

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/950,713 Expired - Fee Related US8697440B2 (en) 2003-10-14 2010-11-19 Compositions and methods for producing gamma-carboxylated proteins

Country Status (28)

Country Link
US (2) US7842477B2 (de)
EP (2) EP2292748B1 (de)
JP (1) JP4658946B2 (de)
KR (1) KR101201020B1 (de)
CN (2) CN1867669B (de)
AR (1) AR046180A1 (de)
AT (1) ATE491789T1 (de)
AU (2) AU2004281350B2 (de)
BR (1) BRPI0415277A (de)
CA (1) CA2541764C (de)
CO (1) CO5690657A2 (de)
DE (1) DE602004030603D1 (de)
DK (1) DK1678297T3 (de)
ES (2) ES2532470T3 (de)
GB (1) GB0324044D0 (de)
IL (2) IL174633A (de)
IS (1) IS8447A (de)
MX (1) MXPA06004060A (de)
MY (1) MY141859A (de)
NO (1) NO339740B1 (de)
NZ (2) NZ578306A (de)
PL (1) PL1678297T3 (de)
PT (1) PT1678297E (de)
RU (2) RU2372401C2 (de)
TW (1) TWI355419B (de)
UY (1) UY28558A1 (de)
WO (1) WO2005038019A1 (de)
ZA (1) ZA200603038B (de)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080312127A1 (en) * 2005-04-13 2008-12-18 Ann Lovgren Host Cell Comprising a Vector for Production of Proteins Requiring Gamma-Carboxylation
US20090047273A1 (en) * 2007-07-06 2009-02-19 Anna Harrysson Method For Production of Recombinant Human Thrombin ['644]
US20100159512A1 (en) * 2006-03-06 2010-06-24 Kurt Osther Method for the preparation of recombinant human thrombin and fibrinogen
US20110092429A1 (en) * 2003-10-14 2011-04-21 Christel Fenge Compositions and methods for producing gamma-carboxylated proteins

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7687233B2 (en) 2003-09-23 2010-03-30 The University Of North Carolina At Chapel Hill Methods and compositions for the correlation of single nucleotide polymorphisms in the vitamin K epoxide reductase gene and warfarin dosage
AU2005329450A1 (en) 2005-03-15 2006-09-28 University Of North Carolina At Chapel Hill Methods and compositions for producing active Vitamin K-dependent proteins
WO2007040162A1 (ja) * 2005-09-30 2007-04-12 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute 培養細胞による組換えヒトトロンビンの製造方法
US7745484B2 (en) 2005-11-21 2010-06-29 Amgen Inc. Beta-secretase modulators and methods of use
JP5526332B2 (ja) * 2005-12-21 2014-06-18 シーエヌジェイ ホールディングス，インコーポレイテッド 組み換え法によって生物活性ビタミンｋ依存性タンパク質を製造する方法
JP6050927B2 (ja) * 2007-04-26 2016-12-21 シーエヌジェイ ホールディングス，インコーポレイテッド 高シアル酸含量を有する組換えビタミンｋ依存性タンパク質およびその調製方法
BR112013008034A2 (pt) * 2010-10-06 2016-06-14 Medimmune Ltd método para normalizar hemeostase comprometida
US9631002B2 (en) 2010-12-21 2017-04-25 The University Of North Carolina At Chapel Hill Methods and compositions for producing active vitamin K-dependent proteins
KR20140057348A (ko) 2011-09-06 2014-05-12 메디뮨 엘엘씨 응고 인자를 처리하는 방법
RU2500816C1 (ru) * 2012-07-19 2013-12-10 Общество с ограниченной ответственностью "Международный биотехнологический центр "Генериум" ("МБЦ "Генериум") РЕКОМБИНАНТНАЯ ПЛАЗМИДНАЯ ДНК рАК380, КОДИРУЮЩАЯ ПОЛИПЕПТИД РЕКОМБИНАНТНОГО ФАКТОРА IХ СВЕРТЫВАЕМОСТИ КРОВИ ЧЕЛОВЕКА, ЛИНИЯ КЛЕТОК Cricetulus griseus CHO 1E6 - ПРОДУЦЕНТ РЕКОМБИНАНТНОГО ФАКТОРА IХ СВЕРТЫВАЕМОСТИ КРОВИ ЧЕЛОВЕКА И СПОСОБ ПОЛУЧЕНИЯ ПОЛИПЕПТИДА, ОБЛАДАЮЩЕГО АКТИВНОСТЬЮ РЕКОМБИНАНТНОГО ФАКТОРА IХ
JP5830486B2 (ja) 2013-03-29 2015-12-09 シスメックス株式会社 組換えバキュロウイルスおよびその利用
EP3083677B1 (de) * 2013-12-20 2019-08-28 Novartis AG Neuartige eukaryotische zellen und verfahren zur rekombinanten expression eines produkts von interesse
CN109207462B (zh) * 2018-08-01 2021-05-11 深圳市新产业生物医学工程股份有限公司 异常凝血酶原及其制备方法和包含其的检测剂、试剂盒及应用

Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0052827A2 (de) 1980-11-21 1982-06-02 BEHRINGWERKE Aktiengesellschaft Verfahren zur Herstellung von Blutgerinnungsfaktoren und danach hergestellte Präparation der Faktoren II und VII
US4599308A (en) * 1981-10-06 1986-07-08 Hamer Dean H Protein from SV40 recombinants
WO1988003926A1 (en) 1986-11-17 1988-06-02 New England Medical Center Enhancing gamma-carboxylation of recombinant vitamin k-dependent proteins
US4784950A (en) * 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
WO1989012685A1 (en) 1987-05-18 1989-12-28 Integrated Genetics, Inc. Improved protein c molecules and method for making and activating same
WO1992001795A1 (en) 1990-07-23 1992-02-06 Zymogenetics, Inc. Gamma-carboxylase and methods of use
US5118614A (en) 1988-01-18 1992-06-02 Tessek Sdruzeni Praha Concentrates of coagulation factors ii, vii, ix and x, method of their preparation and use
US5122458A (en) 1984-08-24 1992-06-16 The Upjohn Company Use of a bgh gdna polyadenylation signal in expression of non-bgh polypeptides in higher eukaryotic cells
WO1992019636A1 (en) 1991-05-08 1992-11-12 The University Of North Carolina At Chapel Hill Vitamin k-dependent carboxylase
EP0607392A1 (de) 1992-07-14 1994-07-27 Alpha Therapeutic Corporation Reinigung von plasmafraktion
EP0700682A2 (de) 1994-08-26 1996-03-13 BEHRINGWERKE Aktiengesellschaft Arzneimittel, enthaltend Prothrombin zur Antagonisierung von Blut-Anticoagulanzien
WO1996034966A2 (en) 1995-05-04 1996-11-07 American Red Cross Engineering protein posttranslational modification in transgenic organisms
US5648254A (en) * 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
US5866122A (en) 1996-03-20 1999-02-02 Immuno Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
WO1999033983A1 (en) 1997-12-24 1999-07-08 Immunex Corporation V201 dna and polypeptides
US5958893A (en) * 1994-03-23 1999-09-28 The University Of Iowa Research Foundation Genes and proteins for treating cystic fibrosis
WO2001004146A2 (en) 1999-07-09 2001-01-18 Cohesion Technologies, Inc. Ecarin polypeptides, polynucleotides encoding ecarin, and methods for use thereof
WO2001007068A1 (en) 1999-07-23 2001-02-01 Case Western Reserve University Novel methods and reagents for the treatment of osteoarthritis
US6224864B1 (en) 1986-12-03 2001-05-01 Pharmacia & Upjohn Company Antibiotic 10381A, and process for the preparation of anitbiotics 10381B
US6342372B1 (en) 1993-09-15 2002-01-29 Chiron Corporation Eukaryotic layered vector initiation systems for production of recombinant proteins
WO2002029045A2 (en) 2000-10-02 2002-04-11 Novo Nordisk A/S Method for the production of vitamin k-dependent proteins
US20020106381A1 (en) * 2000-06-13 2002-08-08 High Katherine A. Methods for administering recombinant adeno-associated virus virions to humans previously exposed to adeno-associated virus
EP1405912A1 (de) 2001-07-06 2004-04-07 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Genetisch modifziertes ecarin und verfahren zu dessen herstellung
EP1405910A1 (de) 2001-07-06 2004-04-07 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Verfahren zur herstelllung von menschlichem thrombin mittels genmodifikationstechnik
EP1407780A1 (de) 2001-07-10 2004-04-14 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Pharmazeutisch stabile hämostatische zusammensetzungen
WO2005030039A2 (en) 2003-09-23 2005-04-07 University Of North Carolina At Chapel Hill Methods and compositions for the correlation of single nucleotide polymorphisms in the vitamin k epoxide reductase gene and warfarin dosage
WO2005038019A1 (en) 2003-10-14 2005-04-28 Astrazeneca Ab Method for producing gamma-carboxylated proteins
WO2005040367A1 (en) 2003-10-14 2005-05-06 Baxter International Inc. Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives
WO2006067116A1 (en) 2004-12-21 2006-06-29 Novo Nordisk Health Care Ag Expression of gamma-carboxylated polypeptides in gamma-carboxylation deficient host systems
EP1676911A1 (de) 2003-10-24 2006-07-05 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Neue rekombinante tierzellen mit hoher proteinproduktion, verfahren zur konstruktion davon sowie verfahren zur massenproduktion von protein unter verwendung davon
WO2006110083A1 (en) 2005-04-13 2006-10-19 Astrazeneca Ab A host cell comprising a vector for production of proteins requiring gamma-carboxylation
WO2007065173A2 (en) 2005-12-02 2007-06-07 Wake Forest University Health Sciences Compositions and methods for increasing production of recombinant gamma-carboxylated proteins
US20080045453A1 (en) 2005-12-21 2008-02-21 Drohan William N Method of producing biologically active vitamin K dependent proteins by recombinant methods
US20090047273A1 (en) 2007-07-06 2009-02-19 Anna Harrysson Method For Production of Recombinant Human Thrombin ['644]

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ZA862768B (en) * 1985-04-17 1986-12-30 Zymogenetics Inc Expression of factor vii and ix activities in mammalian cells
CA2280700A1 (en) * 1997-02-14 1998-08-20 American Red Cross Expression of active human factor ix in mammary tissue of transgenic animals

Patent Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4404187A (en) 1980-11-21 1983-09-13 Behringwerke Aktiengesellschaft Method for rendering factors II and VII hepatitis-safe with a chelating agent
EP0052827A2 (de) 1980-11-21 1982-06-02 BEHRINGWERKE Aktiengesellschaft Verfahren zur Herstellung von Blutgerinnungsfaktoren und danach hergestellte Präparation der Faktoren II und VII
US4599308A (en) * 1981-10-06 1986-07-08 Hamer Dean H Protein from SV40 recombinants
US5122458A (en) 1984-08-24 1992-06-16 The Upjohn Company Use of a bgh gdna polyadenylation signal in expression of non-bgh polypeptides in higher eukaryotic cells
US4784950A (en) * 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
WO1988003926A1 (en) 1986-11-17 1988-06-02 New England Medical Center Enhancing gamma-carboxylation of recombinant vitamin k-dependent proteins
US6224864B1 (en) 1986-12-03 2001-05-01 Pharmacia & Upjohn Company Antibiotic 10381A, and process for the preparation of anitbiotics 10381B
WO1989012685A1 (en) 1987-05-18 1989-12-28 Integrated Genetics, Inc. Improved protein c molecules and method for making and activating same
US5648254A (en) * 1988-01-15 1997-07-15 Zymogenetics, Inc. Co-expression in eukaryotic cells
US5118614A (en) 1988-01-18 1992-06-02 Tessek Sdruzeni Praha Concentrates of coagulation factors ii, vii, ix and x, method of their preparation and use
WO1992001795A1 (en) 1990-07-23 1992-02-06 Zymogenetics, Inc. Gamma-carboxylase and methods of use
US5965789A (en) 1991-01-11 1999-10-12 American Red Cross Engineering protein posttranslational modification by PACE/furin in transgenic non-human mammals
WO1992019636A1 (en) 1991-05-08 1992-11-12 The University Of North Carolina At Chapel Hill Vitamin k-dependent carboxylase
EP0607392A1 (de) 1992-07-14 1994-07-27 Alpha Therapeutic Corporation Reinigung von plasmafraktion
US6342372B1 (en) 1993-09-15 2002-01-29 Chiron Corporation Eukaryotic layered vector initiation systems for production of recombinant proteins
US5958893A (en) * 1994-03-23 1999-09-28 The University Of Iowa Research Foundation Genes and proteins for treating cystic fibrosis
EP0700682A2 (de) 1994-08-26 1996-03-13 BEHRINGWERKE Aktiengesellschaft Arzneimittel, enthaltend Prothrombin zur Antagonisierung von Blut-Anticoagulanzien
WO1996034966A3 (en) 1995-05-04 1996-12-05 American Nat Red Cross Engineering protein posttranslational modification in transgenic organisms
WO1996034966A2 (en) 1995-05-04 1996-11-07 American Red Cross Engineering protein posttranslational modification in transgenic organisms
US6224862B1 (en) 1996-03-20 2001-05-01 Baxter Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
US5866122A (en) 1996-03-20 1999-02-02 Immuno Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
US6039945A (en) 1996-03-20 2000-03-21 Baxter Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
US6165974A (en) 1996-03-20 2000-12-26 Baxter Aktiengesellschaft Pharmaceutical preparation for treating blood coagulation disorders
WO1999033983A1 (en) 1997-12-24 1999-07-08 Immunex Corporation V201 dna and polypeptides
US6413737B1 (en) 1999-07-09 2002-07-02 Cohesion Technologies, Inc. Ecarin prothrombin protease and methods
WO2001004146A2 (en) 1999-07-09 2001-01-18 Cohesion Technologies, Inc. Ecarin polypeptides, polynucleotides encoding ecarin, and methods for use thereof
WO2001007068A1 (en) 1999-07-23 2001-02-01 Case Western Reserve University Novel methods and reagents for the treatment of osteoarthritis
US20020106381A1 (en) * 2000-06-13 2002-08-08 High Katherine A. Methods for administering recombinant adeno-associated virus virions to humans previously exposed to adeno-associated virus
WO2002029083A2 (en) 2000-10-02 2002-04-11 Novo Nordisk A/S Industrial-scale serum-free production of recombinant proteins in mammalian cells
WO2002029045A2 (en) 2000-10-02 2002-04-11 Novo Nordisk A/S Method for the production of vitamin k-dependent proteins
WO2002029083A3 (en) 2000-10-02 2003-08-21 Novo Nordisk As Industrial-scale serum-free production of recombinant proteins in mammalian cells
EP1405912A1 (de) 2001-07-06 2004-04-07 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Genetisch modifziertes ecarin und verfahren zu dessen herstellung
EP1405910A1 (de) 2001-07-06 2004-04-07 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Verfahren zur herstelllung von menschlichem thrombin mittels genmodifikationstechnik
US20040197858A1 (en) 2001-07-06 2004-10-07 Hiroshi Yonemura Process for producing human thrombin by gene modification technique
EP1407780A1 (de) 2001-07-10 2004-04-14 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Pharmazeutisch stabile hämostatische zusammensetzungen
WO2005030039A2 (en) 2003-09-23 2005-04-07 University Of North Carolina At Chapel Hill Methods and compositions for the correlation of single nucleotide polymorphisms in the vitamin k epoxide reductase gene and warfarin dosage
US7482141B2 (en) 2003-09-23 2009-01-27 University Of North Carolina At Chapel Hill Methods and compositions for vitamin K epoxide reductase
WO2005040367A1 (en) 2003-10-14 2005-05-06 Baxter International Inc. Vitamin k epoxide recycling polypeptide vkorc1, a therapeutic target of coumarin and their derivatives
US20050164367A1 (en) 2003-10-14 2005-07-28 Astrazeneca Ab Protein
WO2005038019A1 (en) 2003-10-14 2005-04-28 Astrazeneca Ab Method for producing gamma-carboxylated proteins
EP1676911A1 (de) 2003-10-24 2006-07-05 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Neue rekombinante tierzellen mit hoher proteinproduktion, verfahren zur konstruktion davon sowie verfahren zur massenproduktion von protein unter verwendung davon
WO2006067116A1 (en) 2004-12-21 2006-06-29 Novo Nordisk Health Care Ag Expression of gamma-carboxylated polypeptides in gamma-carboxylation deficient host systems
WO2006110083A1 (en) 2005-04-13 2006-10-19 Astrazeneca Ab A host cell comprising a vector for production of proteins requiring gamma-carboxylation
US20080312127A1 (en) 2005-04-13 2008-12-18 Ann Lovgren Host Cell Comprising a Vector for Production of Proteins Requiring Gamma-Carboxylation
WO2007065173A2 (en) 2005-12-02 2007-06-07 Wake Forest University Health Sciences Compositions and methods for increasing production of recombinant gamma-carboxylated proteins
US20080045453A1 (en) 2005-12-21 2008-02-21 Drohan William N Method of producing biologically active vitamin K dependent proteins by recombinant methods
US20090047273A1 (en) 2007-07-06 2009-02-19 Anna Harrysson Method For Production of Recombinant Human Thrombin ['644]

Non-Patent Citations (107)

* Cited by examiner, † Cited by third party
Title
Amino acid sequence for wild type ecarin (EBI accession No. Q90495). Last modified Jan. 19, 2010, 5 pages.
Bajaj et al. "A Simplified Procedure for Purification of Human Prothrombin, Factor IX and Factor X" Prep. Biochem. 1981 (11) 397-412.
Bajaj et al. "Isolation and Characterization of Human Factor VII. Activation of Factor VII by Factor X" J. Biotechnol. 1981 (256) 253-259.
Bandyopadhyay et al., "gamma-Glutamyl carboxylation: an extracellular posttranslational modification that antedates the divergence of molluscs, anthropods, and chordates," PNAS 99(3):1264-1269 (2002).
Bandyopadhyay et al., "γ-Glutamyl carboxylation: an extracellular posttranslational modification that antedates the divergence of molluscs, anthropods, and chordates," PNAS 99(3):1264-1269 (2002).
Begley et al., "A conserved motif within the vitamin K-dependent carboxylase gene is widely distributed across animal phyla," The Journal of Biological Chemistry 275(46):36245-36249 (2000).
Bentley et al., "Differential Efficiency of Expression of Humanized Antibodies in Transient Transfected Mammalian Cells", Hybridoma, vol. 17(6), pp. 559-567 (1998).
Bishop et al. "Comparison of Recombinant Human Thrombin and Plasma-Derived Human alpha-Thrombin" Sem Throm Hem. 2006 (32) 86-97.
Bishop et al. "Comparison of Recombinant Human Thrombin and Plasma-Derived Human α-Thrombin" Sem Throm Hem. 2006 (32) 86-97.
Broun et al. "Catalytic Plasticity of Fatty Acid Modification Enzymes Underlying Chemical Diversity of Plant Lipids" Science. 1998 (282) 1315-1317.
Camire et al., "Enhanced gamma-Carboxylation of Recombinant Factor X Using a Chimeric Construct Containing the Prothrombin Propeptide", Biochemistry, vol. 39, pp. 14322-14329 (2000).
Camire et al., "Enhanced γ-Carboxylation of Recombinant Factor X Using a Chimeric Construct Containing the Prothrombin Propeptide", Biochemistry, vol. 39, pp. 14322-14329 (2000).
Clark et al. "The Secreted Protein Discovery Initiative (SPDI), a Large-Scale Effort to Identify Novel Human Secreted and Transmembrane Proteins: A Bioinformatics Assessment" Genome Res. 2003 (13) 2265-2270.
Cote et al., "Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A and R284A)," The Journal of Biological Chemistry 269(15):11374-11380 (1994).
Czerwiec et al., "Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile," Eur. J. Biochem 269:6162-6172 (2002).
Czerwiec et al., "Expression and characterization of recombinant vitamin K-dependent γ-glutamyl carboxylase from an invertebrate, Conus textile," Eur. J. Biochem 269:6162-6172 (2002).
Fair et al. "Biosynthesis and Secretion of Factor VII, Protein C, Protein S, and the Protein C Inhibitor From a Human Hepatoma Cell Line" Blood. 1986 (67) 64-70.
Falkner et al. "High Level Expression of Active Human Prothrombin in a Vaccine Virus Expression System" Thrombosis and Haemostasis. 1992 (68) 119-124.
Fischer et al., "purification of recombinant human coagulation factors II and IX and protein S expressed in recombinant Vaccinia virus-infected Vero cells," Journal of Biotechnology 38:129-136 (1995).
Fish & Richardson P.C., Amendment in Reply to Restriction Requirement dated Apr. 6, 2009 in U.S. Appl. No. 12/167,614, filed Aug. 4, 2009, 9 pages.
Fish & Richardson P.C., Response to Restriction Requirement and Preliminary Amendment dated Apr. 2, 2009 in U.S. Appl. No. 11/572,870, filed Sep. 25, 2009, 16 pages.
Gamma Glutamyl Carboxylase. UniPro Database. [online], [retrieved on Jan. 14, 2010] Retrieved from the UniPro Database using Internet .
Gamma Glutamyl Carboxylase. UniPro Database. [online], [retrieved on Jan. 14, 2010] Retrieved from the UniPro Database using Internet <URL: http://www.uniprot.org/uniprot/?query=gamma+glutamyl+carboxylase&sort=score>.
Gustafsson et al., "Codon bias and heterologous protein expression" Trends in Biotechnol., 22(7):346-353 (2004).
Hallgren et al. "Carboxylase overexpression effects full carboxylation but poor release and secretion of a factor IX: implications for the release of vitamin K-dependent proteins," Biochemistry 41:15045-15055 (2002).
Harvey et al, "Mutagenesis of the gamma-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site," The Journal of Biological Chemistry 278(10):8363-8369 (2003).
Harvey et al, "Mutagenesis of the γ-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site," The Journal of Biological Chemistry 278(10):8363-8369 (2003).
Hellstern "Production and Composition of Prothrombin Complex Concentrates: Correlation between Composition and Therapeutic Efficiency" Thrombosis Research. 1999 (95) S7-S12.
Hellstern et al. "Prothrombin Complex Concentrates: Indications, Contraindications, and Risks: A Task Force Summary" Thrombosis Research. 1999 (95) S3-S6.
Hellstern et al. Preface Thrombosis Research. 1999 (95) S1.
Herlitschka et al., "Overexpression of Human Prothrombin in Permanent Cell Lines Using a Dominant Selection/Amplification Fusion Marker", Protein Expression and Purification, vol. 8, pp. 358-364 (1996).
Herlitschka et al., "Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker," Protein Expression and Purification 8:358-364 (1996).
Himmelspach et al. "A Fully Recombinant Partial Prothrombin Complex Effectively Bypasses fVII In Vitro and In Vivo" Thromb Haemost. 2002 (88) 1003-1011.
Jorgensen et al., "Expression of completely gamma-carboxylated recombinant human prothrombin," The Journal of Biological Chemistry 262(14):6729-6734 (1987).
Jorgensen et al., "Expression of completely γ-carboxylated recombinant human prothrombin," The Journal of Biological Chemistry 262(14):6729-6734 (1987).
Kaufman et al. "Expression, Purification, and Characterization of Recombinant gamma-Carboxylated Factor IX Synthesized in Chinese Hamster Ovary Cells" J. Biol. Chem. 1986 (261) 9622-9628.
Kaufman et al. "Expression, Purification, and Characterization of Recombinant γ-Carboxylated Factor IX Synthesized in Chinese Hamster Ovary Cells" J. Biol. Chem. 1986 (261) 9622-9628.
K�hler "Thrombogenicity of Prothrombin Complex Concentrates" Thrombosis Research. 1999 (95) S13-S17.
Kini et al. "The intriguing world of prothrombin activators from snake venom" Toxicon. 2005 (45) 1133-1145.
Köhler "Thrombogenicity of Prothrombin Complex Concentrates" Thrombosis Research. 1999 (95) S13-S17.
Koresawa et al., "Synthesis of a new cre recombinase gene based on optimal codon usage for mammalian systems" J. Biochem., 127:367-372 (2000).
Kozak, "An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs", Nucleic Acids Research, vol. 15(20), pp. 8125-8148 (1987).
Kozak, "An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs", Nucleic Acids Research, vol. 15(20), pp. 8125-8148 (1987).
Kozak, "Downstream Secondary Structure Facilitates Recognition of Intiator Codons by Eukaryotic Ribosomes", Proceedings of the National Academy of Sciences of the United States of America, vol. 87(21), pp. 8301-8305 (1990).
Kozak, "Point Mutations Define a Sequence Flanking the AUG Initiator Codon That Modulates Translation by Eukaryotic Ribosomes", Cell, vol. 44, pp. 283-292 (1986).
Li et al., "Identification of the gene for vitamin K epoxide reductase", Nature 427:541-544 (2004).
Lingenfelter et al., "Isolation of the Human gamma-Carboxylase and a gamma-Carboxylase-Associated Protein from Factor IX-Expressing Mammalian Cells", Biochemistry, vol. 35, pp. 8234-8243 (1996).
Lingenfelter et al., "Isolation of the Human γ-Carboxylase and a γ-Carboxylase-Associated Protein from Factor IX-Expressing Mammalian Cells", Biochemistry, vol. 35, pp. 8234-8243 (1996).
Lucas et al. "High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector" Nucleic Acids Research. 1996 (24) 1774-1779.
Malhotra et al., "The kinetics of activation of normal and gamma-carboxyglutamic acid-deficient prothrombins," The Journal of Biological Chemistry 260(1):279-287 (1985).
Malhotra et al., "The kinetics of activation of normal and γ-carboxyglutamic acid-deficient prothrombins," The Journal of Biological Chemistry 260(1):279-287 (1985).
McCawley et al., "Matrix metalloproteinases: they're not just for matrix anymore!" Curr. Opinion Cell Biol., 13:534-540 (2001).
Melcher et al., "Plasmid vectors with a 5 -hybrid intron facilitate high-level glycoprotein expression in CHO-cells", Biochimica et Biophysica Acta 1575, pp. 49-53 (2002).
Melcher et al., "Plasmid vectors with a 5 -hybrid intron facilitate high-level glycoprotein expression in CHO-cells", Biochimica et Biophysica Acta 1575, pp. 49-53 (2002).
Munns et al. "Vitamin K-dependent synthesis and modification of precursor prothrombin in cultured H-35 hepatoma cells" Proc. Natl. Acad. Sci. 1976 (73) 2803-2807.
Newby A.C., "Matrix metalloproteinases regulate migration, proliferation, and death . . . " Cardiovascular Res., 69:614-624 (2006).
Nishida et al. "cDNA cloning and deduced amino acid sequence of prothrombin activator (ecarin) from Kenyan Echis carinatus venom" Biochem. 1995 (34) 1771-1778.
Nucleotide sequence of human prothrombin (EBI accession No. AJ972449). Last modified Oct. 21, 2008, 8 pages.
PCT Written Opinion for Application No. PCT/SE2008/050836, dated Jan. 21, 2010, 10 pages.
PCT/SE2004/001453 International Search Report Jan. 21, 2005.
Pei et al. "Expression, isolation, and characterization of an active site (serine 528-alanine) mutant of recombinant bovine prothrombin" J. Biol. Chem. 1991 (266) 9598-9604.
Pejler et al. "Thrombin Is Inactivated by Mast Cell Secretory Granule Chymase" J. Biol. Chem. 1993 (268) 11817-11822.
Rehemtulla et al., "In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells," Proc. Natl. Acad. Sci. USA 90:4611-4615 (1993).
Rehemtulla et al., "In vitro and in vivo functional characterization of bovine vitamin K-dependent γ-carboxylase expressed in Chinese hamster ovary cells," Proc. Natl. Acad. Sci. USA 90:4611-4615 (1993).
Restriction Requirement in U.S. Appl. No. 11/572,870, mailed Apr. 2, 2009, 5 pages.
Restriction Requirement in U.S. Appl. No. 12/167,614, mailed Apr. 6, 2009, 8 pages.
Robertson "Genes Encoding Vitamin-K Epoxide Reductase Are Present in Drosophila and Trypanosomatid Protists" Genetics. 2004 (168) 1077-1080.
Roddie et al., "Haemostasis and thrombosis-Recombinant coagulation factors," Blood Reviews 11:169-177 (1997).
Rost et al. "Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2" Nature. 2004 (427) 537-541.
Rouet et al., "A Potent Enhancer Made of Clustered Liver-specified Elements in the Transcription Control Sequences of Human alpha1-Microglobulin/Bikunin Gene*", The Journal of Biological Chemistry, vol. 267(29), pp. 20765-20773 (1992).
Rouet et al., "A Potent Enhancer Made of Clustered Liver-specified Elements in the Transcription Control Sequences of Human α1-Microglobulin/Bikunin Gene*", The Journal of Biological Chemistry, vol. 267(29), pp. 20765-20773 (1992).
Russo et al., "Biologically active recombinant prothrombin and antithrombin III expressed in a human hepatoma/vaccinia virus system," Biotechnology and Applied Biochemistry 14:222-223 (1991).
Russo et al., "Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin," Protein Expression and Purification 10:214-225 (1997).
Sadler "K is for koagulation" Nature. 2004 (427) 493-494.
Scharrer "The Need for Highly Purified Products to Treat Hemophilia B" Acta Haematol. 1995 (94) 2-7.
Scharrer et al., "Products used to treat hemophilia: evolution of treatment for hemophilia A and B," in: Lee et al., eds., Textbook of Hemophilia (New York, Blackwell, 2005), Ch. 23, pp. v-x and 131-135.
Seffernick et al. "Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different" J. Bacteriol. 2001 (183) 2405-2410.
Slimko et al. "Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels" J. Neuroscience Methods. 2003 (124) 75-81.
Stanley et al., "The propeptides of the vitamin K-dependent proteins possess different affinities for the vitamin K-dependent carboxylase," The Journal of Biological Chemistry 274(24):16940-16944 (1999).
Strausberg et al. "Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences" Proc. Natl. Acad. Sci. U.S.A. 2002 (99) 16899-16903.
Sun et al., "Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X," Blood 106 (12):3811-3815 (2005).
Tans et al. "Prothrombin Activation by Snake Venom Proteases" J. Toxicol.-Toxin Reviews. 1993 (12) 155-173.
Uma�a et al., "Tetracycline-Regulated Overexpression of Glycosyltransferases in Chinese Hamster Ovary Cells", Biotechnology and Bioengineering 65, pp. 542-549 (1999).
Umaña et al., "Tetracycline-Regulated Overexpression of Glycosyltransferases in Chinese Hamster Ovary Cells", Biotechnology and Bioengineering 65, pp. 542-549 (1999).
USPTO Non-Final Office Action in U.S. Appl. No. 12/167,614, mailed Sep. 29, 2009, 19 pages.
Vo et al., "Undercarboxylation of recombinant prothrombin revealed by analysis of gamma-carboxyglutamic acid using capillary electrophoresis and laser-induced fluorescence," Febs Letters 445:256-260 (1999).
Vo et al., "Undercarboxylation of recombinant prothrombin revealed by analysis of γ-carboxyglutamic acid using capillary electrophoresis and laser-induced fluorescence," Febs Letters 445:256-260 (1999).
Wajih et al. "The Inhibitory Effect of Calumenin on the Vitamin K-dependent gamma-Carboxylation System" J. Biol. Chem. 2004 (279) 25276-25283.
Wajih et al. "The Inhibitory Effect of Calumenin on the Vitamin K-dependent γ-Carboxylation System" J. Biol. Chem. 2004 (279) 25276-25283.
Wajih et al., "Engineering of a Recombinant Vitamin K-dependent gamma-Carboxylation System with Enhanced gamma-Carboxyglutamic Acid Forming Capacity," J. Biol. Chem. 280:10540-10547 (2005).
Wajih et al., "Engineering of a Recombinant Vitamin K-dependent γ-Carboxylation System with Enhanced γ-Carboxyglutamic Acid Forming Capacity," J. Biol. Chem. 280:10540-10547 (2005).
Wajih et al., "Increased Production of Functional Recombinant Human Clotting Factor IX by Baby Hamster Kidney Cells Engineered to Overexpress VKORC1, the Vitamin K 2,3-Expoxide-reducing Enzyme of the Vitamin K Cycle," J. Biol. Chem. 280:31603-31607 (2005).
Walker et al., "On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems," The Journal of Biological Chemistry 276(11):7769-7774 (2001).
Walker et al., "On a potential global role for vitamin K-dependent γ-carboxylation in animal systems," The Journal of Biological Chemistry 276(11):7769-7774 (2001).
Wallin et al. "Vitamin K 2,3-epoxide reductase and the vitamin K-dependent gamma-carboxylation system" Thrombosis Research. 2003 (108) 221-226.
Wallin et al. "Vitamin K 2,3-epoxide reductase and the vitamin K-dependent γ-carboxylation system" Thrombosis Research. 2003 (108) 221-226.
Wallin et al. "Vitamin K-dependent Carboxylation and Vitamin K Metabolism in Liver" J. Clin. Invest. 1985 (76) 1879-1884.
Wang et al. "The Growth Inhibitory Effects of Vitamins K and Their Actions on Gene Expression" Hepatology. 1995 (22) 876-881.
Whisstock et al. "Prediction of protein function from protein sequence and structure" Q. Rev. Biophysics. 2003 (36) 307-340.
Witkowski et al. "Conversion of beta-Ketoacyl Synthase to a Malonyl Decarboxylase by Replacement of the Active-Site Cysteine with Glutamine" Biochemistry. 1999 (38) 11643-11650.
Witkowski et al. "Conversion of β-Ketoacyl Synthase to a Malonyl Decarboxylase by Replacement of the Active-Site Cysteine with Glutamine" Biochemistry. 1999 (38) 11643-11650.
Wu et al., "Cloning and expression of the cDNA for human gamma-glutamyl carboxylase," Science 254:1634-1636 (1991).
Wu et al., "Cloning and expression of the cDNA for human γ-glutamyl carboxylase," Science 254:1634-1636 (1991).
Wu et al., "N-Glycosylation contributes to the intracellular stability of prothrombin precursors in the endoplasmic reticulum," Thrombosis Research 96:91-98 (1999).
Yonemura et al. "Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin" J. Biochem. 2004 (135) 577-582.
Yonemura et al. "Preparation of recombinant α-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin" J. Biochem. 2004 (135) 577-582.
Zhang et al., "Relative Promoter Strengths in Four Human Prostate Cancer Cell Lines Evaluated by Particle Bombardment-Mediated Gene Transfer," The Prostate, 51:286-292 (2002).

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110092429A1 (en) * 2003-10-14 2011-04-21 Christel Fenge Compositions and methods for producing gamma-carboxylated proteins
US8697440B2 (en) * 2003-10-14 2014-04-15 Medimmune Limited Compositions and methods for producing gamma-carboxylated proteins
US20080312127A1 (en) * 2005-04-13 2008-12-18 Ann Lovgren Host Cell Comprising a Vector for Production of Proteins Requiring Gamma-Carboxylation
US7989193B2 (en) 2005-04-13 2011-08-02 Medimmune Limited Compositions and methods for producing gamma-carboxylated proteins
US8304224B2 (en) 2005-04-13 2012-11-06 Medimmune Limited Compositions and methods relating to proteins requiring gamma-carboxylation
US20100159512A1 (en) * 2006-03-06 2010-06-24 Kurt Osther Method for the preparation of recombinant human thrombin and fibrinogen
US20090047273A1 (en) * 2007-07-06 2009-02-19 Anna Harrysson Method For Production of Recombinant Human Thrombin ['644]
US8206967B2 (en) 2007-07-06 2012-06-26 Medimmune Limited Method for production of recombinant human thrombin

Also Published As

Publication number Publication date
US8697440B2 (en) 2014-04-15
CN102443548B (zh) 2014-03-12
CA2541764A1 (en) 2005-04-28
ES2357603T3 (es) 2011-04-28
NO20061470L (no) 2006-05-12
JP4658946B2 (ja) 2011-03-23
ZA200603038B (en) 2007-09-26
RU2006110147A (ru) 2007-11-20
KR101201020B1 (ko) 2012-11-14
US20050164367A1 (en) 2005-07-28
PL1678297T3 (pl) 2011-11-30
CN1867669B (zh) 2011-10-19
AU2008205445A1 (en) 2008-09-04
KR20060123152A (ko) 2006-12-01
NO339740B1 (no) 2017-01-30
RU2372401C2 (ru) 2009-11-10
MY141859A (en) 2010-07-16
MXPA06004060A (es) 2006-06-28
EP1678297A1 (de) 2006-07-12
DK1678297T3 (da) 2011-03-14
NZ578306A (en) 2011-02-25
TWI355419B (en) 2012-01-01
WO2005038019A1 (en) 2005-04-28
JP2007508820A (ja) 2007-04-12
DE602004030603D1 (de) 2011-01-27
TW200523365A (en) 2005-07-16
AU2004281350B2 (en) 2008-05-15
EP1678297B1 (de) 2010-12-15
IL174633A (en) 2013-06-27
ATE491789T1 (de) 2011-01-15
IS8447A (is) 2006-05-10
HK1155475A1 (en) 2012-05-18
IL226313A0 (en) 2013-06-27
GB0324044D0 (en) 2003-11-19
CN102443548A (zh) 2012-05-09
AR046180A1 (es) 2005-11-30
CN1867669A (zh) 2006-11-22
RU2009123931A (ru) 2010-12-27
PT1678297E (pt) 2011-03-15
AU2004281350A1 (en) 2005-04-28
UY28558A1 (es) 2005-05-31
CA2541764C (en) 2015-03-31
EP2292748A1 (de) 2011-03-09
BRPI0415277A (pt) 2006-12-19
NZ546584A (en) 2009-08-28
IL174633A0 (en) 2006-08-20
CO5690657A2 (es) 2006-10-31
ES2532470T3 (es) 2015-03-27
US20110092429A1 (en) 2011-04-21
EP2292748B1 (de) 2014-12-17

Similar Documents

Publication Publication Date Title
US8697440B2 (en) Compositions and methods for producing gamma-carboxylated proteins
US8304224B2 (en) Compositions and methods relating to proteins requiring gamma-carboxylation
AU2008275828B2 (en) Method for production of recombinant human thrombin `644
HK1155475B (en) Method for producing gamma-carboxylated proteins

Legal Events

Date Code Title Description
AS Assignment

Owner name: ASTRAZENECA AB, SWEDEN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FENGE, CHRISTEL;LOVGREN, ANN;THELIN, ANDERS;REEL/FRAME:015690/0398;SIGNING DATES FROM 20040903 TO 20040909

Owner name: ASTRAZENECA AB, SWEDEN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FENGE, CHRISTEL;LOVGREN, ANN;THELIN, ANDERS;SIGNING DATES FROM 20040903 TO 20040909;REEL/FRAME:015690/0398

AS Assignment

Owner name: MEDIMMUNE LIMITED, UNITED KINGDOM

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ASTRAZENECA AB;REEL/FRAME:025788/0442

Effective date: 20110208

CC Certificate of correction
FPAY Fee payment

Year of fee payment: 4

SULP Surcharge for late payment
FEPP Fee payment procedure

Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.)

LAPS Lapse for failure to pay maintenance fees

Free format text: PATENT EXPIRED FOR FAILURE TO PAY MAINTENANCE FEES (ORIGINAL EVENT CODE: EXP.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

STCH Information on status: patent discontinuation

Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362

FP Lapsed due to failure to pay maintenance fee

Effective date: 20181130